Methods for treating disorders of calcium homeostasis

ABSTRACT

The present invention provides methods for using tricyclic amide compounds, such as  
                 
 
     to treat disorders of calcium homeostasis. Such disorders include familial benign hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism and renal secondary hyperparathyroidism.

[0001] This application claims the benefit of U.S. Provisional PatentApplication No. 60/414,948, filed Sep. 30, 2002, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for treating calciumhomeostatic disorders in a subject.

BACKGROUND OF THE INVENTION

[0003] The complex interplay between calcitropic hormones and theirCa²⁺-translocating target tissues is crucial for maintaining theextracellular/serum calcium concentration in humans within its normalrange. These hormones include, inter alia, the parathyroid hormone(PTH); others include calcitonin and 1,25-dihydroxyvitamin D₃. Theactions of these hormones regulate renal Ca²⁺ reabsorption, intestinalCa²⁺ absorption and skeletal Ca²⁺ mobilization. A key element in thisprocess is a G-protein coupled receptor (GPCR) termed the Calcium IonSensing Receptor (CaSR). Human CaSR was originally cloned from theparathyroid gland and was subsequently identified in several othertissues including the kidney (Aida, et al., (1995) Biochem. Biophys.Res. Comm. 214:524-529 and Garrett, et al., (1995) J. Biol. Chem.270:12919-12925). Furthermore, CaSR was identified in several speciesincluding X. laevis, rabbits, rats and cows (Brown, et al., (1993)Nature 366:575-580; Riccardi, et al., (1995) Proc. Natl. Acad. Sci.92:131-135 and Butters, et al., (1997) J. Bone Mineral Res. 12:568-579).CaSR may regulate serum calcium levels by modulating the level ofsynthesis and secretion of PTH and by modulating the extent of Ca²⁺reabsorption in the kidneys (Chattopadhyay, (2000) Int. J. Biochem. CellBio. 32:789-804; Chattopadhyay, et al., (2000) Cellular Signal.12:361-366).

[0004] An example of a calcium homeostatic disorder is familialhypocalciuric hypercalcemia (FHH), which is sometimes called familialbenign hypercalcemia (FBH) or familial benign hypocalciurichypercalcemia (FBHH). Subjects with FHH suffer from lifelong mild tomoderate hypercalcemia. As those with FHH age, they can develop calciumdeposits in their cartilage and suffer bouts of acute pancreatitis (Vanhaeften, et al., (1994) Neth. J. Med. 45(3):110-113; Davies, et al.,(1981) Br. Med. J. (Clin. Res. Ed.) 282(6269): 1023-1025).

[0005] Neonatal severe hyperparathyroidism (NSHPT) is a condition whichcan lead to marked bony demineralization, multiple fractures and ribcage malformation. This condition can be fatal if a parathyroidectomy isnot carried out within the first few weeks of life (Chattopadhyay, etal., (1996) Endocrin. Rev. 17(4): 289-307). NSHPT may be linked todecreased CaSR activity which, in some cases, may be associated with aCaSR mutation (Chattopadhyay, et al., (1996) Endocrine Rev. 17: 289-307;Heath, et al., (1996) J. Clin. Endocrin. Metab. 81:1312-1317; Pearce, etal., (1995) J. Clin. Invest. 96:2683-2692 and Pollak, et al., (1993)Cell 75: 1297-1303).

[0006] Renal secondary hyperparathyroidism is characterized by an excessof serum PTH which is a secondary effect caused by renal failure.Subjects with renal secondary hyperparathyroidism can suffer fromdisabling skeletal diseases such as renal osteodystrophy (Sherrard, etal., (1993) Kidney Int. 43:436-442; Torres, et al., (1995) Kidney Int.47: 1434-1442; Moniere-Faugere, et al., (1996) Nephrol. Dial.Transplant. 11: 111-120).

[0007] One of the most common causes of hypercalcemia is malignancy(i.e., malignancy associated hypercalcemia (MAH) or humoralhypercalcemia of malignancy (HHM)). It is estimated that 10%-20% ofcancer patients suffer from hypercalcemia. The most common cancers thatare associated with the development of MAH are squamous cell lungcancer, squamous cell head and neck cancers, breast cancer, multiplemyeloma, T-cell lymphomas, renal cell cancer and ovarian cancer. Aproximal cause of MAH is believed to be increased bone resorption.Subjects suffering from MAH can suffer from nausea, vomiting, lethargy,confusion and, eventually, death.

[0008] Parathyroid hormone has been known since the 1930s to havecatabolic effects in bone (e.g., causing resorption of calcium from boneto serum). One medical condition which is characterized by increasedbone resorption and increased frequency of bone fractures isosteoporosis. Excess serum parathyroid hormone is likely to be anexacerbating factor in osteoporosis. Thorsen, et al., (1997) (Surgery122(5):882-887) demonstrated an improvement in bone density inpostmenopausal women with hyperparathyroidism after parathyroidectomy.

[0009] Subjects suffering from any of the above-mentioned conditions(e.g., FHH, NSHPT, renal secondary hyperparathyroidism, MAH, HHM orosteoporosis) may benefit from a therapy which leads to a decrease inserum Ca²⁺ and/or serum parathyroid hormone levels. The presentinvention provides, inter alia, methods for treating disorders ofcalcium homeostasis in a subject, such as those discussed above, byadministering a tricyclic amide compound of the invention. Tricyclicamides, such as SCH66336, have been described previously (Liu, et al.,(1998) Cancer Res. 58:4974-4956; Njoroge, et al., (1998) J. Med. Chem.41:1561-1567; U.S. Pat. No. 5,719,148; U.S. Pat. No. 5,874,442; PCTPublication No. WO95/10516) and found to inhibit Farnesyl ProteinTransferase (FPT) mediated farnesylation of the Ras protein and, thus,to be useful for treating malignancy.

[0010] The use of a farnesylation inhibitor, B-i1086 (Nagasu, et al.,(1995) Cancer Res. 55:5310-5314), to treat malignancy-associatedhypercalcemia (MAH) has been studied previously (Aklilu, et al., (1997)Cancer Res. 57:4517-4522). Parathyroid Hormone Related Peptide (PTHRP)has been identified as a pathogenic factor in MAH (Moseley, et al.,(1987) Proc. Natl. Acad. Sci. USA 84:5048-5052; Stewart, et al., (1987)Biochem. Biophys. Res. Comm. 146:672-678; Stewler, et al., (1987) J.Clin. Invest. 80:1803-1807; Rabbani, et al., (1986) Endocrinology118:1200-1210; Li, et al., (1994) Cancer Res. 53:2980-2986) and Akliluet al. found Ras to enhance PTHRP production in rat 3T3 cells. Treatmentof mice bearing Ras-3T3 tumors and suffering from hypercalcemia (MAH),with B- 1086, resulted in a normalization of serum calcium and reductionof serum PTHRP levels. B-1086, however, is a peptidomimetic inhibitorwhich is chemically unrelated to tricyclic amides such as SCH66336. Theeffect of B-1086 on serum PTH levels was not investigated.

SUMMARY OF THE INVENTION

[0011] The present invention provides a method for treating orpreventing disorders of calcium homeostasis (e.g., hypercalcemia) in asubject comprising administering, to the subject, a compound accordingto the following Formula A:

[0012] wherein R1 can be hydrogen (H) or halogen (e.g., F, Cl or Br) R2,R3 and R4 are independently selected from H and halogen (e.g., F, Cl orBr) provided that at least one of R2, R3 and R4 is H;

[0013] “—” represents an optional bond;

[0014] R5 represents C when the optional bond to R5 is present andrepresents CH or N when the optional bond to R5 is absent;

[0015] R6 is selected from

[0016] Preferably, the compound is Formula 1 (see infra). Medicalconditions which may be treated by the methods of the present inventioninclude familial benign hypocalciuric hypercalcemia, neonatal severeprimary hyperparathyroidism and renal secondary hyperparathyroidism.

[0017] The present invention also includes methods for agonizing theCaSR, or for decreasing the level of Ca²⁺, PTH or PTHrP in the serum ofa subject by administering a tricyclic amide compound, for example,comprising formula A, preferably comprising any of formulas 1-81, morepreferably formula 1.

[0018] The tricyclic amide compounds of the present invention may beadministered in association with a second substance for treating orpreventing a calcium homeostatic disorder (e.g., AMG073, NPS467, NPS568,gadolinium, lanthanum, neomycin, Mg²⁺, 1,25-dihydroxyvitamin D,calcitrol, paricalcitrol, doxercalciferol, zoledronic acid, calcitonin,alfacalcidol or oxacalcitriol).

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention provides, generally, methods for treatingdisorders of calcium homeostasis (e.g., hypercalcemia). Specifically,the present invention provides methods for treating calcium homeostaticdisorders by administering a tricyclic amide of the invention (e.g.,Formula 1) to a subject in need of such treatment. Calcium homeostaticdisorders which may be treated by administering the tricyclic amides ofthe present invention (e.g., Formula 1) include disorders associatedwith excess serum Ca²⁺ and/or PTH and/or with an excess mobilization ofCa²⁺ from the bones. Such disorders include, but are by no means limitedto, familial benign hypocalciuric hypercalcemia, neonatal severe primaryhyperparathyroidism, renal secondary hyperparathyroidism, osteoporosis,malignancy-associated hypercalcemia (MAH) and humoral hypercalcemia ofmalignancy (HHM). Without being bound by a single theory, some calciumhomeostatic disorders (e.g., hypercalcemia) may be caused by, forexample, an excess of serum PTH, or PTHrP or a lack of expression oractivity of the CaSR. Again, without being bound by a single theory, thetricyclic amides of the present invention (e.g., Formula 1) may treatcalcium homeostasis disorders (e.g., hypercalcemia) by, for example,agonizing the CaSR (e.g., CaSR in the parathyroid gland or in thekidney) which may, in turn, lead to a decrease in serum PTH or PTHrP.The present invention is not limited by any particular mechanism bywhich a tricyclic amide of the invention (e.g., Formula 1) may treat orprevent a calcium homeostatic disorder.

[0020] The term “Calcium Ion Sensing Receptor”, “Calcium SensingReceptor” or “CaSR” refers to a receptor which is commonly known in theart. A typical human CaSR amino acid sequence is set forth in SEQ ID NO:1 as well as under Genbank Accession No. U20759, U20760 and D50855(Garrett, et al., (1995) J. Biol. Chem. 270(21): 12919-12925; Aida, etal., (1995) Biochem. Biophys. Res. Comm. 214(2):524-529). A typical ratCaSR amino acid sequence is set forth in SEQ ID NO: 2 as well as underGenbank Accession No. U10354 (Riccardi, et al., (1995) Proc. Natl. Acad.Sci. USA 92 (1):131-135). The term includes receptors from any species,preferably mammalian species (e.g., rat, mouse, monkey, rabbit, cow) andmost preferably from humans.

[0021] The term “parathyroid hormone” or “PTH” is commonly known in theart and refers to the hormone secreted by the parathyroid gland. Atypical, mature, human PTH is described by Varicek, et al., (1983) Proc.Natl. Acad. Sci. 80 (8), 2127-2131 and Hendy, et al., (1981) Proc. Natl.Acad. Sci. 78(12): 7365-7369). A typical, mature, rat PTH is describedby Schmelzer, et al., (1987) Nucleic Acids Res. 15(16): 6740. The termincludes PTH from any species, preferably mammalian species (e.g., dog,cat, pig, rat, mouse, monkey, rabbit, cow) and most preferably fromhumans.

[0022] “PTHrP” refers to hormone, parathyroid hormone related protein,secreted by tumor cells which produces effects similar to those of PTH(see, for example, Suva et al., (1987) Science 237 (4817):893-896).

[0023] The term “subject” includes any organism, preferably a mammal(e.g., dog, cat, pig, rat, mouse, monkey, rabbit, cow) and, mostpreferably, a human.

[0024] The term “compound” includes small molecules (e.g., tricyclicamides, polymers, organic molecules, hydrocarbons, inorganic ions andsalts), proteins (e.g., antibodies, oligopeptides, polypeptides,hormones and enzymes), saccharides (e.g., monosaccharides,oligosaccharides and polysaccharides) and nucleic acids (e.g.,oligonucleotides, polynucleotides, genes, plasmids, DNA and RNA).

[0025] The term “e.g. ” means “exempli gratia” or “for example” and, ingeneral, precedes one or more non-limiting examples.

Tricyclic Amides

[0026] Disorders of calcium homeostasis, in a subject, may be treated byadministering a tricyclic amide compound to the subject. Preferably, thetricyclic amide compound comprises a formula set forth in U.S. Pat. No.5,719,148 or in U.S. Pat. No. 5,874,442 which are herein incorporated byreference in their entireties. Preferably, the tricyclic amide compoundsof the present invention include the following Formula A:

[0027] wherein R1 can be hydrogen (H) or halogen (e.g., F, Cl or Br)

[0028] R2, R3 and R4 are independently selected from H and halogen(e.g., F, Cl or Br) provided that at least one of R2, R3 and R4 is H;

[0029] “—” an optional bond;

[0030] R5 represents C when the optional bond to R5 is present andrepresents CH or N when the optional bond to R5 is absent;

[0031] R6 is selected from

[0032] Preferably the optional bonds between C5 and C6 and on C11 (toR5), in Formula A, are absent. Preferably, R1 is halogen, morepreferably Br. Representative compounds of Formula A include thosewherein:

[0033] (1) R1 is halogen (preferably Br), R2 is halogen (preferably Br),R3 is halogen (preferably Cl), and R4 is H; or wherein

[0034] (2) R1 is halogen (preferably Br), R2 is H, R3 is halogen(preferably Cl) and R4 is halogen (preferably Br).

[0035] Preferably, the tricyclic amide is a compound of Formula Awherein R1 is halogen (preferably Br), R2 is H, R3 is halogen(preferably Cl) and R4 is halogen (preferably Br), the optional bondbetween C5 and C6 and to R5 is absent, R5 is CH and R6 is

[0036] Most preferably the tricyclic amide is Formula 1:

[0037] Those skilled in the art will appreciate that the tricyclic ringsystem is numbered according to the following illustration:

[0038] Other representative examples of the tricyclic amides of thepresent invention include but are by no means limited to:

Pharmaceutical Compositions, Dosage and Administration

[0039] The present invention includes pharmaceutical compositionscomprising the tricyclic amide compounds of the invention (e.g.,Formula 1) along with a pharmaceutically acceptable carrier.

[0040] The pharmaceutical compositions can be adapted for any mode ofadministration e.g., for oral, parenteral, e.g., subcutaneous (“SC”),intramuscular (“IM”), and intraperitoneal (“IP”) or topicaladministration or by inhalation (e.g., orally or intranasally).Preferably, tricyclic amide compounds of the invention (e.g., Formula 1)are administered orally (e.g., in a pill, capsule or tablet).

[0041] Such pharmaceutical compositions may be formulated by combiningthe tricyclic amide compounds (e.g., Formula 1) or an equivalent amountof a pharmaceutically acceptable salt thereof with a suitable, inert,pharmaceutically acceptable carrier or diluent that may be either solidor liquid.

[0042] Acceptable solid form preparations include powders, tablets,dispersible granules, capsules, cachets and suppositories. The powdersand tablets may be comprised of, for example, from about 1 to about 95percent active ingredient. Suitable solid carriers are known in the art,e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose.Tablets, powders, cachets and capsules can be used as solid dosage formssuitable for oral administration. Examples of pharmaceuticallyacceptable carriers and methods of manufacture for various compositionsmay be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences,18^(th) Edition, (1990), Mack Publishing Co.; Easton, Pa.

[0043] Liquid form preparations include solutions, suspensions andemulsions. Such preparations include, for example, water orwater-propylene glycol solutions for parenteral injection. Solid formpreparations may be converted into liquid preparations shortly beforeuse for either oral or administration. Parenteral forms to be injectedintravenously, intramuscularly or subcutaneously are usually in the formof sterile solutions and may contain tonicity agents (e.g., salts orglucose), and buffers. Opacifiers may be included in oral solutions,suspensions and emulsions. Liquid form preparations may also includesolutions for intranasal administration.

[0044] Aerosol preparations suitable for inhalation may includesolutions and solids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas,e.g., nitrogen.

[0045] Also included are solid form preparations which are intended tobe converted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,syrups, suspensions and emulsions.

[0046] Preferably, the pharmaceutical preparation is in a unit dosageform. In such form, the preparation is subdivided into suitably sizedunit doses containing appropriate quantities of the active component,e.g., an effective amount to achieve the desired purpose.

[0047] In general, normal total serum calcium levels range from about8.6 mg/dl to about 10.3 mg/dl (adult human), normal serum intact PTHlevels range from about 10 pg/ml to about 65 pg/ml (adult human) andnormal serum ionized Ca²⁺ levels range from about 4.64 to about 5.28mg/dl (adult human) (Tietz Fundamentals of Clinical Chemistry (5^(th)Ed); W. B. Saunders Co.; New York; 2001). Methods for measuring serumCa²⁺ or PTH are commonly known in the art. Although it is preferable forthe methods of the present invention for treating calcium homeostaticdisorders to lead to normal total serum Ca²⁺ and/or serum ionized Ca²⁺and/or PTH levels in a subject, the present invention is not so limited.The present invention includes methods for treating calcium homeostaticdisorders which lead to any detectable change in total serum Ca²⁺ levelsor serum ionized Ca²⁺ levels or serum PTH levels toward a normal rangeor which lead to any degree of alleviation of symptoms associated withthe particular disorder of calcium homeostasis.

Combinations

[0048] The tricyclic amide compounds of the present invention may alsobe administered to a subject in association with a known, secondsubstance which is useful for treating disorders of calcium homeostasis.Such substances include, but are not limited to, AMG073 (Goodman, etal., (2002) J. Am. Soc. Nephrol. 13:1017-1024 ), NPS467 (Nemeth, et al.,(1998) Proc. Natl. Acad. Sci. 95:4040-4045), NPS568 (Nemeth, et al.,(1998) Proc. Natl. Acad. Sci. 95:4040-4045), gadolinium, lanthanum,neomycin, Mg²⁺, 1,25-dihydroxyvitamin D (Delmez, et al., (1989) J. Clin.Invest. 83:1349-1355), calcitrol, paricalcitrol (Martin, et al., (1998)J. Am. Soc. Nephrol. 9:1427-1432), doxercalciferol (Frazao, et al.,(2000) Am. J. Kidney Dis. 36:562-565), zoledronic acid (e.g., Zometa;Davidson, (2001) Am. J. Health Syst. Pharm. 58 Suppl. 3:S8-15),calcitonin, alfacalcidol and oxacalcitriol. Each of these substances arewell known in the art.

[0049] The tricyclic amide compounds of the invention may be formulatedwith the second substance into a single composition or into two or moreseparate compositions for simultaneous consumption. Alternatively, atricyclic amide compound of the invention may be administered to asubject at a different time than when the second substance isadministered; for example, each administration may be givennon-simultaneously at several intervals over a given period of time.

EXAMPLES Example 1 Effect of the Compound of Formula 1 on CalciumHomeostasis and PTH

[0050] A study was undertaken to assess the potential nephrotoxicity ofthe compound of Formula 1 in rats. The rats were 6 week old femalesweighing 120.7 to 173.5 g at dosing initiation.

[0051] The study design is summarized below in Tables 1-3: TABLE 1One-Month Exploratory Nephrotoxicity Study of the Compound of Formula 1in Female Rats: Study Design. Dose No. Rats Total Daily Dose SuspensionNo. Bled for Duration of Test/control Dose Volume Conc. Female PlasmaDosing Group Article (mg/kg) (mg/kg) (mg/kg) Rats Analysis¹ (days)Control Methyl- 0 5 0 20 20² 3, 7, 14 or cellulose 30 (0.4%; aqueous)Test Formula 1 180 5 36 20 20² 3, 7, 14 or 30

[0052] TABLE 2 One-Month Exploratory Nephrotoxicity Study of theCompound of Formula 1 in Female Rats: Observations and Measurements.Investigation Performed Investigation Performed Viability and Dailybeginning Urinalysis/Urine Days 2, 6, 13 and 29 Clinical Week-1Chemistry Observations Body Weight Weekly beginning Organ Weights¹ Days2, 6, 13 and 29 Week-1, and days of randomization and terminal sacrificeFood Consumption Weekly beginning Necropsy Days 2, 6, 13 and 29 Week-1(Macroscopic Observations) Water Consumption Days 2, 6, 13 and 29Histopathology Days 2, 6, 13 and 29 (Microscopic Observations)² Plasmaanalysis for Days 2, 6, 13 and 29 Ultrastructural Days 13 and 29 Formula1 (2 hours post-dose) Pathology³ Serum Chemistry Days 2, 6, 13 and 29

[0053] TABLE 3 One-Month Exploratory Nephrotoxicity Study of theCompound of Formula 1 in Female Rats: Assignment of Rats for PlasmaAnalysis, Serum Chemistry, Urinalysis, Urine Chemistry andHistopathology Evaluations. Animal Blood (ml- Urinalysis/ Order/Group/Water site of Plasma Serum Urine Histo- Sacrifice Day Consumptionbleeding) Analysis (ml)¹ Chemistry Chemistry pathology  1^(st) ✓ 4/aorta1.5 ADH Std² —  2^(nd) ✓ 4/aorta 1.5 ADH Std² —  3^(rd) ✓ 4/aorta 1.5ADH Std² —  4^(th) ✓ 4/aorta 1.5 ADH Std² —  5^(th) ✓ 4/aorta 1.5 ADHStd² —  6^(th) ✓ 4/aorta — std, PTH, Std² K, PT vitDs, free Ca  7^(th) ✓4/aorta — std, PTH, Std³ K, PT vitDs, free Ca  8^(th) ✓ 4/aorta — std,PTH, Std³ K, PT vitDs, free Ca  9^(th) ✓ 4/aorta — std, PTH, Std³ K, PTvitDs, free Ca 10^(th) ✓ 4/aorta — std, PTH, Std³ K, PT vitDs, free Ca#tube and the following parameters were measured: hemolysis; lipemia;icterus; glucose; urea nitrogen; creatinine; alkaline phosphatase; totalprotein; albumin; globulin (calculated); albumin/globulin ratio(calculated); sodium; potassium; chloride; calcium (total); freecalcium; phosphorus; parathyroid hormone; vitamin D; antidiuretichormone (ADH); 1, 25-dihydroxy vitamin D; 25-hydroxy vitamin D.#analysis. Unused voided samples were combined with the respective24-hour samples for urinalysis. The following parameters were measuredin freshly voided samples: color; clarity; pH; protein; glucose;ketones; bilirubin; blood; urobilinogen; microscopic analysis. For24-hour samples: sodium; potassium; chloride; calcium (total);phosphorus; creatinine clearance; volume; osmolality.

[0054] TABLE 4 Mean calcium and phosphorus excretion (g/kg/24 hours).Day Day Day Day Day Day Day Day 2 2 6 6 13 13 29 29 Formula 1 Ca P Ca PCa P Ca P  0 mg/kg 7.30 57.93 5.55 51.60 9.56 37.8 10.88 39.43 180 mg/kg5.65 55.86 32.99 13.54 20.69 12.11 19.24 7.30

[0055] TABLE 5 Mean serum PTH (ng/ml). Formula 1 Day 2 Day 6 Day 13 Day29  0 mg/kg 94 54 89 90 180 mg/kg 60 51 68 44

[0056] TABLE 6 Mean serum ADH (pg/ml). Formula 1 Day 2 Day 6 Day 13 Day29  0 mg/kg 193 97 115 619  180 mg/kg 350 513 547 616¹

[0057] TABLE 7 Mean urine volume (ml/kg/24 hours) and osmolality(mOsm/kg). Day Day Day Day Day Day Day Day 2 2 6 6 13 13 29 29 Formula 1Vol. Osmo Vol. Osmo Vol. Osmo Vol. Osmo  0 mg/kg 75 1541 71 1573 1001176 84 1073 180 mg/kg 124 646 195 646 177 655 166 606

[0058] Within 6 days of dosing at 180 mg/kg, calcium excretion hadincreased in the urine and phosphorus excretion was decreased.Furthermore, a decrease in serum PTH was observed in rats dosed with thecompound of Formula 1 and, within 28 days, 5 out of 5 rats evaluated hadlower PTH serum concentrations than did the control rats. Decreases inurine osmolality, increases in urine volume and increases inantidiuretic hormone (ADH) concentrations suggest the development ofnephrogenic diabetes insipidus (failure of kidneys to concentrate urinein response to ADH).

[0059] These data also strongly suggest that structurally relatedtricyclic amide compounds (e.g., formulas 2-81or those disclosed in U.S.Pat. No. 5,719,148 or in U.S. Pat. No. 5,874,442) would exhibit in vivoproperties which are similar to those described above for the compoundof Formula 1. Specifically, the structurally related tricyclic amidecompounds should also be useful for treating calcium homeostaticdisorders such as hypercalcemia.

[0060] Without being bound by a single theory, agonism of the CaSR inthe parathyroid gland of a subject (e.g., by administration ofFormula 1) may lead to lower serum parathyroid hormone levels which inturn may lead to lower Ca²⁺ serum levels. The parathyroid cells maydecrease PTH secretion by fusing secretory granules containing PTH withlysosomes; this would lead to PTH degradation. Again, without beingbound by a single theory, at the kidney level, the Calcium Ion SensingReceptor may help regulate calcium ion reabsorption. Calcium regulationat the kidney may be independent of parathyroid hormone (Brown, et al.,(1995) N. Engl. J. Med. 333(4):234-240). Under this theory, when theCalcium Ion Sensing Receptor is activated by increased calcium ions inthe plasma, there is a diminished uptake of calcium from the filtrate,resulting in increased calciuria.

Example 2 Micrographic Analysis of Rat Parathyroid Glands

[0061] Microscopic evaluation of the parathyroid glands of rats dosedwith 180 mg/kg Formula 1 revealed cytoplasmic vacuolation.

[0062] Electron photomicrographs of the right parathyroid gland from twocontrol rats sacrificed on Day 13 (Nos. 26F and 29F), two control ratssacrificed on Day 29 (Nos. 39F and 40F) and two Formula 1-dosed ratssacrificed on Day 29 (Nos. 76F and 77F) were examined forultrastructural changes. The majority of parathyroid chief cells fromthe two Formula 1-dosed rats sacrificed on Day 29 had less conspicuouscell borders with fewer, less distinctive cytoplasmic projections. Thissuggests that the gland from which the cells were obtained was lessactive in PTH production/secretion. Consistent with the lightmicroscopic appearance, the cytoplasm contained numerous, generallymembrane-bound, vesicles, approximately 0.2-3 μm in diameter, that weregenerally electron lucent with occasional irregular, variablyelectron-dense material. The number of myeloid bodies and fat globuleswas minimally increased. A single rounded parathyroid epithelial cell inanimal No. 77F appeared apoptotic with condensed chromatin and intactmitochondria and contained numerous vesicles similar to those previouslydescribed. The apoptotic cell is consistent with the single cellnecrosis observed at the light microscopic level. This too suggests thatthe gland from which the cells were obtained was less active in PTHproduction/secretion.

[0063] Considering the increased number of vesicles and myeloid bodiesin the Formula 1-dosed rats, some vesicles may have fused with lysosomes(crinophagy) which would lead to a decrease in PTH secretion. There wasno evidence of exhaustion vacuoles. Additionally, there were no changesindicating chronic stimulation such as increased rough endoplasmicreticulum and prominent Golgi apparatus.

[0064] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

[0065] Patents, patent applications, publications, Genbank submissionaccession numbers, product descriptions, and protocols are citedthroughout this application, the disclosures of which are incorporatedherein by reference in their entireties for all purposes.

1 2 1 1088 PRT Homo sapiens VARIANT (537)..(546) deleted in humancalcium ion sensing receptor variant 1 Met Ala Phe Tyr Ser Cys Cys TrpVal Leu Leu Ala Leu Thr Trp His 1 5 10 15 Thr Ser Ala Tyr Gly Pro AspGln Arg Ala Gln Lys Lys Gly Asp Ile 20 25 30 Ile Leu Gly Gly Leu Phe ProIle His Phe Gly Val Ala Ala Lys Asp 35 40 45 Gln Asp Leu Lys Ser Arg ProGlu Ser Val Glu Cys Ile Arg Tyr Asn 50 55 60 Phe Arg Gly Phe Arg Trp LeuGln Ala Met Ile Phe Ala Ile Glu Glu 65 70 75 80 Ile Asn Ser Ser Pro AlaLeu Leu Pro Asn Leu Thr Leu Gly Tyr Arg 85 90 95 Ile Phe Asp Thr Cys AsnThr Val Ser Lys Ala Leu Glu Ala Thr Leu 100 105 110 Ser Phe Val Ala GlnAsn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe 115 120 125 Cys Asn Cys SerGlu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala 130 135 140 Thr Gly SerGly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe 145 150 155 160 TyrIle Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165 170 175Lys Asn Gln Phe Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His 180 185190 Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp 195200 205 Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu210 215 220 Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp PheSer 225 230 235 240 Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile GlnHis Val Val 245 250 255 Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile ValVal Phe Ser Ser 260 265 270 Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu IleVal Arg Arg Asn Ile 275 280 285 Thr Gly Lys Ile Trp Leu Ala Ser Glu AlaTrp Ala Ser Ser Ser Leu 290 295 300 Ile Ala Met Pro Gln Tyr Phe His ValVal Gly Gly Thr Ile Gly Phe 305 310 315 320 Ala Leu Lys Ala Gly Gln IlePro Gly Phe Arg Glu Phe Leu Lys Lys 325 330 335 Val His Pro Arg Lys SerVal His Asn Gly Phe Ala Lys Glu Phe Trp 340 345 350 Glu Glu Thr Phe AsnCys His Leu Gln Glu Gly Ala Lys Gly Pro Leu 355 360 365 Pro Val Asp ThrPhe Leu Arg Gly His Glu Glu Ser Gly Asp Arg Phe 370 375 380 Ser Asn SerSer Thr Ala Phe Arg Pro Leu Cys Thr Gly Asp Glu Asn 385 390 395 400 IleSer Ser Val Glu Thr Pro Tyr Ile Asp Tyr Thr His Leu Arg Ile 405 410 415Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln 420 425430 Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe Thr Asn Gly Ser 435440 445 Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gln Val Leu Lys His Leu450 455 460 Arg His Leu Asn Phe Thr Asn Asn Met Gly Glu Gln Val Thr PheAsp 465 470 475 480 Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile AsnTrp His Leu 485 490 495 Ser Pro Glu Asp Gly Ser Ile Val Phe Lys Glu ValGly Tyr Tyr Asn 500 505 510 Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe IleAsn Glu Glu Lys Ile 515 520 525 Leu Trp Ser Gly Phe Ser Arg Glu Pro LeuThr Phe Val Leu Ser Val 530 535 540 Leu Gln Val Pro Phe Ser Asn Cys SerArg Asp Cys Leu Ala Gly Thr 545 550 555 560 Arg Lys Gly Ile Ile Glu GlyGlu Pro Thr Cys Cys Phe Glu Cys Val 565 570 575 Glu Cys Pro Asp Gly GluTyr Ser Asp Glu Thr Asp Ala Ser Ala Cys 580 585 590 Asn Lys Cys Pro AspAsp Phe Trp Ser Asn Glu Asn His Thr Ser Cys 595 600 605 Ile Ala Lys GluIle Glu Phe Leu Ser Trp Thr Glu Pro Phe Gly Ile 610 615 620 Ala Leu ThrLeu Phe Ala Val Leu Gly Ile Phe Leu Thr Ala Phe Val 625 630 635 640 LeuGly Val Phe Ile Lys Phe Arg Asn Thr Pro Ile Val Lys Ala Thr 645 650 655Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe Ser Leu Leu Cys Cys Phe 660 665670 Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro Gln Asp Trp Thr Cys Arg 675680 685 Leu Arg Gln Pro Ala Phe Gly Ile Ser Phe Val Leu Cys Ile Ser Cys690 695 700 Ile Leu Val Lys Thr Asn Arg Val Leu Leu Val Phe Glu Ala LysIle 705 710 715 720 Pro Thr Ser Phe His Arg Lys Trp Trp Gly Leu Asn LeuGln Phe Leu 725 730 735 Leu Val Phe Leu Cys Thr Phe Met Gln Ile Val IleCys Val Ile Trp 740 745 750 Leu Tyr Thr Ala Pro Pro Ser Ser Tyr Arg AsnGln Glu Leu Glu Asp 755 760 765 Glu Ile Ile Phe Ile Thr Cys His Glu GlySer Leu Met Ala Leu Gly 770 775 780 Phe Leu Ile Gly Tyr Thr Cys Leu LeuAla Ala Ile Cys Phe Phe Phe 785 790 795 800 Ala Phe Lys Ser Arg Lys LeuPro Glu Asn Phe Asn Glu Ala Lys Phe 805 810 815 Ile Thr Phe Ser Met LeuIle Phe Phe Ile Val Trp Ile Ser Phe Ile 820 825 830 Pro Ala Tyr Ala SerThr Tyr Gly Lys Phe Val Ser Ala Val Glu Val 835 840 845 Ile Ala Ile LeuAla Ala Ser Phe Gly Leu Leu Ala Cys Ile Phe Phe 850 855 860 Asn Lys IleTyr Ile Ile Leu Phe Lys Pro Ser Arg Asn Thr Ile Glu 865 870 875 880 GluVal Arg Cys Ser Thr Ala Ala His Ala Phe Lys Val Ala Ala Arg 885 890 895Ala Thr Leu Arg Arg Ser Asn Val Ser Arg Lys Arg Ser Ser Ser Leu 900 905910 Gly Gly Ser Thr Gly Ser Thr Pro Ser Ser Ser Ile Ser Ser Lys Ser 915920 925 Asn Ser Glu Asp Pro Phe Pro Arg Pro Glu Arg Gln Lys Gln Gln Gln930 935 940 Pro Leu Ala Leu Thr Gln Gln Glu Gln Gln Gln Gln Pro Leu ThrLeu 945 950 955 960 Pro Gln Gln Gln Arg Ser Gln Gln Gln Pro Arg Cys LysGln Lys Val 965 970 975 Ile Phe Gly Ser Gly Thr Val Thr Phe Ser Leu SerPhe Asp Glu Pro 980 985 990 Gln Lys Asn Ala Met Ala His Arg Asn Ser ThrHis Gln Asn Ser Leu 995 1000 1005 Glu Ala Gln Lys Ser Ser Asp Thr LeuThr Arg His Gln Pro Leu 1010 1015 1020 Leu Pro Leu Gln Cys Gly Glu ThrAsp Leu Asp Leu Thr Val Gln 1025 1030 1035 Glu Thr Gly Leu Gln Gly ProVal Gly Gly Asp Gln Arg Pro Glu 1040 1045 1050 Val Glu Asp Pro Glu GluLeu Ser Pro Ala Leu Val Val Ser Ser 1055 1060 1065 Ser Gln Ser Phe ValIle Ser Gly Gly Gly Ser Thr Val Thr Glu 1070 1075 1080 Asn Val Val AsnSer 1085 2 1079 PRT Rattus norvegicus 2 Met Ala Ser Tyr Ser Cys Cys LeuAla Leu Leu Ala Leu Ala Trp His 1 5 10 15 Ser Ser Ala Tyr Gly Pro AspGln Arg Ala Gln Lys Lys Gly Asp Ile 20 25 30 Ile Leu Gly Gly Leu Phe ProIle His Phe Gly Val Ala Ala Lys Asp 35 40 45 Gln Asp Leu Lys Ser Arg ProGlu Ser Val Glu Cys Ile Arg Tyr Asn 50 55 60 Phe Arg Gly Phe Arg Trp LeuGln Ala Met Ile Phe Ala Ile Glu Glu 65 70 75 80 Ile Asn Ser Ser Pro SerLeu Leu Pro Asn Met Thr Leu Gly Tyr Arg 85 90 95 Ile Phe Asp Thr Cys AsnThr Val Ser Lys Ala Leu Glu Ala Thr Leu 100 105 110 Ser Phe Val Ala GlnAsn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe 115 120 125 Cys Asn Cys SerGlu His Ile Pro Ser Thr Ile Ala Val Val Gly Ala 130 135 140 Thr Gly SerGly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Phe 145 150 155 160 TyrIle Pro Gln Val Ser Tyr Ala Ser Ser Ser Arg Leu Leu Ser Asn 165 170 175Lys Asn Gln Tyr Lys Ser Phe Leu Arg Thr Ile Pro Asn Asp Glu His 180 185190 Gln Ala Thr Ala Met Ala Asp Ile Ile Glu Tyr Phe Arg Trp Asn Trp 195200 205 Val Gly Thr Ile Ala Ala Asp Asp Asp Tyr Gly Arg Pro Gly Ile Glu210 215 220 Lys Phe Arg Glu Glu Ala Glu Glu Arg Asp Ile Cys Ile Asp PheSer 225 230 235 240 Glu Leu Ile Ser Gln Tyr Ser Asp Glu Glu Glu Ile GlnGln Val Val 245 250 255 Glu Val Ile Gln Asn Ser Thr Ala Lys Val Ile ValVal Phe Ser Ser 260 265 270 Gly Pro Asp Leu Glu Pro Leu Ile Lys Glu IleVal Arg Arg Asn Ile 275 280 285 Thr Gly Arg Ile Trp Leu Ala Ser Glu AlaTrp Ala Ser Ser Ser Leu 290 295 300 Ile Ala Met Pro Glu Tyr Phe His ValVal Gly Gly Thr Ile Gly Phe 305 310 315 320 Gly Leu Lys Ala Gly Gln IlePro Gly Phe Arg Glu Phe Leu Gln Lys 325 330 335 Val His Pro Arg Lys SerVal His Asn Gly Phe Ala Lys Glu Phe Trp 340 345 350 Glu Glu Thr Phe AsnCys His Leu Gln Glu Gly Ala Lys Gly Pro Leu 355 360 365 Pro Val Asp ThrPhe Val Arg Ser His Glu Glu Gly Gly Asn Arg Leu 370 375 380 Leu Asn SerSer Thr Ala Phe Arg Pro Leu Cys Thr Gly Asp Glu Asn 385 390 395 400 IleAsn Ser Val Glu Thr Pro Tyr Met Asp Tyr Glu His Leu Arg Ile 405 410 415Ser Tyr Asn Val Tyr Leu Ala Val Tyr Ser Ile Ala His Ala Leu Gln 420 425430 Asp Ile Tyr Thr Cys Leu Pro Gly Arg Gly Leu Phe Thr Asn Gly Ser 435440 445 Cys Ala Asp Ile Lys Lys Val Glu Ala Trp Gln Val Leu Lys His Leu450 455 460 Arg His Leu Asn Phe Thr Asn Asn Met Gly Glu Gln Val Thr PheAsp 465 470 475 480 Glu Cys Gly Asp Leu Val Gly Asn Tyr Ser Ile Ile AsnTrp His Leu 485 490 495 Ser Pro Glu Asp Gly Ser Ile Val Phe Lys Glu ValGly Tyr Tyr Asn 500 505 510 Val Tyr Ala Lys Lys Gly Glu Arg Leu Phe IleAsn Glu Glu Lys Ile 515 520 525 Leu Trp Ser Gly Phe Ser Arg Glu Val ProPhe Ser Asn Cys Ser Arg 530 535 540 Asp Cys Gln Ala Gly Thr Arg Lys GlyIle Ile Glu Gly Glu Pro Thr 545 550 555 560 Cys Cys Phe Glu Cys Val GluCys Pro Asp Gly Glu Tyr Ser Gly Glu 565 570 575 Thr Asp Ala Ser Ala CysAsp Lys Cys Pro Asp Asp Phe Trp Ser Asn 580 585 590 Glu Asn His Thr SerCys Ile Ala Lys Glu Ile Glu Phe Leu Ala Trp 595 600 605 Thr Glu Pro PheGly Ile Ala Leu Thr Leu Phe Ala Val Leu Gly Ile 610 615 620 Phe Leu ThrAla Phe Val Leu Gly Val Phe Ile Lys Phe Arg Asn Thr 625 630 635 640 ProIle Val Lys Ala Thr Asn Arg Glu Leu Ser Tyr Leu Leu Leu Phe 645 650 655Ser Leu Leu Cys Cys Phe Ser Ser Ser Leu Phe Phe Ile Gly Glu Pro 660 665670 Gln Asp Trp Thr Cys Arg Leu Arg Gln Pro Ala Phe Gly Ile Ser Phe 675680 685 Val Leu Cys Ile Ser Cys Ile Leu Val Lys Thr Asn Arg Val Leu Leu690 695 700 Val Phe Glu Ala Lys Ile Pro Thr Ser Phe His Arg Lys Trp TrpGly 705 710 715 720 Leu Asn Leu Gln Phe Leu Leu Val Phe Leu Cys Thr PheMet Gln Ile 725 730 735 Leu Ile Cys Ile Ile Trp Leu Tyr Thr Ala Pro ProSer Ser Tyr Arg 740 745 750 Asn His Glu Leu Glu Asp Glu Ile Ile Phe IleThr Cys His Glu Gly 755 760 765 Ser Leu Met Ala Leu Gly Ser Leu Ile GlyTyr Thr Cys Leu Leu Ala 770 775 780 Ala Ile Cys Phe Phe Phe Ala Phe LysSer Arg Lys Leu Pro Glu Asn 785 790 795 800 Phe Asn Glu Ala Lys Phe IleThr Phe Ser Met Leu Ile Phe Phe Ile 805 810 815 Val Trp Ile Ser Phe IlePro Ala Tyr Ala Ser Thr Tyr Gly Lys Phe 820 825 830 Val Ser Ala Val GluVal Ile Ala Ile Leu Ala Ala Ser Phe Gly Leu 835 840 845 Leu Ala Cys IlePhe Phe Asn Lys Val Tyr Ile Ile Leu Phe Lys Pro 850 855 860 Ser Arg AsnThr Ile Glu Glu Val Arg Ser Ser Thr Ala Ala His Ala 865 870 875 880 PheLys Val Ala Ala Arg Ala Thr Leu Arg Arg Pro Asn Ile Ser Arg 885 890 895Lys Arg Ser Ser Ser Leu Gly Gly Ser Thr Gly Ser Ile Pro Ser Ser 900 905910 Ser Ile Ser Ser Lys Ser Asn Ser Glu Asp Arg Phe Pro Gln Pro Glu 915920 925 Arg Gln Lys Gln Gln Gln Pro Leu Ser Leu Thr Gln Gln Glu Gln Gln930 935 940 Gln Gln Pro Leu Thr Leu His Pro Gln Gln Gln Gln Gln Pro GlnGln 945 950 955 960 Pro Arg Cys Lys Gln Lys Val Ile Phe Gly Ser Gly ThrVal Thr Phe 965 970 975 Ser Leu Ser Phe Asp Glu Pro Gln Lys Asn Ala MetAla His Arg Asn 980 985 990 Ser Met Arg Gln Asn Ser Leu Glu Ala Gln ArgSer Asn Asp Thr Leu 995 1000 1005 Gly Arg His Gln Ala Leu Leu Pro LeuGln Cys Ala Asp Ala Asp 1010 1015 1020 Ser Glu Met Thr Ile Gln Glu ThrGly Leu Gln Gly Pro Met Val 1025 1030 1035 Gly Asp His Gln Pro Glu MetGlu Ser Ser Asp Glu Met Ser Pro 1040 1045 1050 Ala Leu Val Met Ser ThrSer Arg Ser Phe Val Ile Ser Gly Gly 1055 1060 1065 Gly Ser Ser Val ThrGlu Asn Val Leu His Ser 1070 1075

We claim:
 1. A method for treating or preventing a disorder of calcium homeostasis in a subject comprising administering, to the subject, a compound according to the following formula:

wherein R1 can be hydrogen or halogen R2, R3 and R4 are independently selected from H and halogen provided that at least one of R2, R3 and R4 is H; — represents an optional bond; R5 represents C when the optional bond to R5 is present and represents CH or N when the optional bond to R5 is absent; and R6 is selected from


2. The method of claim 1 wherein the compound of Formula A comprises a formula selected from the group consisting of:


3. A method for treating or preventing a disorder of calcium homeostasis in a subject comprising administering, to the subject, a compound according to the following formula:


4. The method of claim 1 wherein the medical condition is selected from familial benign hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism, renal secondary hyperparathyroidism and osteoporosis.
 5. The method of claim 1 wherein the disorder is mediated by a low level of Calcium Ion Sensing Receptor activity or expression or by excess serum parathyroid hormone.
 6. The method of claim 1 wherein the subject is a human, a dog or a cat.
 7. The method of claim 1 wherein the compound is administered in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.
 8. The method of claim 7 wherein the pharmaceutical composition is in the form of a pill, tablet or capsule.
 9. The method of claim 1 wherein the compound of Formula A is administered in association with a second compound for treating a disorder of calcium homeostasis.
 10. The method of claim 9 wherein the second compound is selected from AMG073, NPS467, NPS568, gadolinium, lanthanum, neomycin, Mg²⁺, 1,25-dihydroxyvitamin D, calcitrol, paricalcitrol, doxercalciferol, zoledronic acid, calcitonin, alfacalcidol and oxacalcitriol.
 11. The method of claim 9 wherein the second compound is administered in combination with a pharmaceutically acceptable carrier in a pharmaceutical composition.
 12. The method of claim 11 wherein said compound of Formula A and said second compound are administered in combination with a pharmaceutically acceptable carrier in a single pharmaceutical composition.
 13. The method of claim 11 wherein said compound of Formula A and said second compound are administered in combination with pharmaceutically acceptable carriers in two separate pharmaceutical compositions.
 14. The method of claim 13 wherein said compound of Formula A and said second compound are administered simultaneously.
 15. The method of claim 13 wherein said compound of Formula A and said second compound are administered non-simultaneously.
 16. The method of claim 11 wherein the pharmaceutical composition is in the form of a pill, tablet or capsule.
 17. A method for decreasing the level of Ca²⁺, parathyroid hormone or parathyroid hormone related protein in the serum of a subject comprising administering a compound according to the following formula to the subject:

wherein R1 can be hydrogen or halogen R2, R3 and R4 are independently selected from H and halogen provided that at least one of R2, R3 and R4 is H; — represents an optional bond; R5 represents C when the optional bond to R5 is present and represents CH or N when the optional bond to R5 is absent; and R6 is selected from


18. A method for treating or preventing a disorder of calcium homeostasis in a subject comprising administering, to the subject, a compound according to the following formula: 